During culture of mature rat hepatocytes as monolayers, c-myc mRNA was found to be expressed transiently within 2 h, decreasing rapidly to the basal level at 10 h. Then its level increased again to over 10-fold the basal level at 24 h, and remained at this high level during culture. The increase of c-myc mRNA in the second phase was shown by nuclear run-off experiments to be due to an increase of its transcription. The second, but not the first, increase in c-myc expression was inversely proportional to the cell density in culture. The expression of c-myc mRNA was not affected by various hormones including growth factors. These results indicate that hepatocytes in culture at lower cell density tend to move from the Go phase to the G1 phase, but remain in the Go phase when cultured at high cell density.